CN112010966B

CN112010966B - Monoclonal antibody aiming at non-RBD (radial basis function) region of new coronavirus spike protein and application thereof

Info

Publication number: CN112010966B
Application number: CN202010926558.8A
Authority: CN
Inventors: 董金华; 李海梅; 陈丽梅
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2020-05-15
Filing date: 2020-09-04
Publication date: 2021-03-19
Anticipated expiration: 2040-09-04
Also published as: CN112010965A; CN111995678B; CN111995676B; CN111961133B; CN112010966A; CN111995678A; CN111995675B; CN111995677B; CN111995675A; CN112010965B; CN111995677A; CN111995676A; CN111961133A

Abstract

A monoclonal antibody and its application against new coronavirus spike protein non-RBD area, this monoclonal antibody binds with specificity of new coronavirus spike protein RBD area, include the complementarity determining region CDRH1 of the heavy chain variable region, CDRH2, CDRH3 and complementarity determining region CDRL1, CDRL2, CDRL3 of the light chain variable region; the monoclonal antibody aiming at the non-RBD region of the spinous process protein of the new coronavirus has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the region outside the RBD region of the spinous process protein on the surface of the SARS-CoV-2 of the new coronavirus, is used for qualitative or quantitative detection of the new coronavirus, can neutralize and weaken certain toxicity of the new coronavirus, and plays a role in preventing or/and treating pneumonia of the new coronavirus.

Description

Monoclonal antibody aiming at non-RBD (radial basis function) region of new coronavirus spike protein and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to the technical field of cellular immunology and molecular biology, and more particularly to a monoclonal antibody aiming at a non-RBD region of a new coronavirus spike protein and application thereof.

Background

The envelope structure of the novel coronavirus comprises three proteins: spinous process protein (Spike protein, S protein), Membrane protein (M protein) and Envelope protein (E protein), the M protein participates in the formation and budding process of the virus Envelope; HE protein constitutes a short bulge of the envelope, possibly associated with early adsorption of coronaviruses, and some HE proteins of coronaviruses cause erythrocyte agglutination and erythrocyte adsorption; the S protein plays a key role in recognizing and combining host cell surface receptors and mediating fusion of virus envelopes and cell membranes. In many mammals, the S protein is cleaved by furin or other enzymes to S1 and S2, where S1 has a receptor attachment site and S2 exhibits mainly fusion activity. The novel coronaviruses can bind to various cellular receptors, of which angiotensin converting enzyme 2 (ACE 2), which is a peptidase, is one of the receptors of cell surface coronaviruses. A part of the region within S1 that binds tightly to ACE2, which we refer to as the Receptor Binding Domain (RBD), is a key element of virus-receptor interaction and is the most variable part of the coronavirus genome. Research shows that cross-species infection can occur due to variation of several amino acids in the RBD, and the RBD contains important virus neutralizing epitopes and is very critical to improving immune antibody response.

The spinous process protein plays a role in the combination of virus and host cell membrane receptor and membrane fusion, and is an important action site of host neutralizing antibody and a key target of vaccine design. The spinous process protein of SARS-CoV-2 (2019-nCoV) interacts with human ACE2 to infect respiratory epithelial cells in humans.

Antibodies are important glycoprotein molecules in the mammalian immune system. The molecular structure is composed of two Heavy chains (Heavy chain) and two Light chains (Light chain), wherein the Heavy chains are divided into Variable regions (VH) and three Constant regions (Constant regions of Heavy chain; CH1, CH2, CH3), and the Light chains are composed of a Variable region (VL) and a Constant region (CL). The variable region is capable of binding specifically to an antigen and is different from one antibody to another, while the constant region of an antibody is determined by the species and subtype of the antibody. The heavy chain variable region of the antibody comprises three Complementarity determining regions (CDRH 1, CDRH2, CDRH 3) and the light chain variable region also comprises three Complementarity determining regions (CDRL 1, CDRL2, CDRL 3), wherein the Complementarity determining regions are also called hypervariable regions and are directly combined with antigen and epitope.

At present, no effective medicine is available for treating the new coronary pneumonia, the effect is not obvious when the conventional antibiotic medicine is adopted for treating, and some medicines have serious toxic and side effects. On the other hand, since antibody drugs play an important role in the treatment of infectious diseases, autoimmune diseases, and the like, and are more targeted, the development of monoclonal antibodies against specific novel coronaviruses is of great importance.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a monoclonal antibody aiming at a non-RBD region of a novel coronavirus spike protein and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention discloses a monoclonal antibody aiming at a non-RBD region of spike protein of SARS-CoV-2 of a new coronavirus, which is specifically combined with the non-RBD region of spike glycoprotein of SARS-CoV-2 of the new coronavirus, and comprises complementarity determining regions CDRH1, CDRH2, CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8.

a preferred monoclonal antibody aiming at the non-RBD region of the spinous process protein of the novel coronavirus SARS-CoV-2 is named D4, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO: 5.

the invention also includes an isolated nucleic acid molecule encoding a monoclonal antibody as described in any one of the above.

The invention also includes an expression vector comprising a nucleic acid molecule as described above, which expression vector comprises, in addition to the nucleic acid molecule as described above, an expression control sequence operably linked to the sequence of said nucleic acid molecule.

An expression vector refers to a nucleic acid vehicle into which a polynucleotide encoding the D4 antibody can be inserted and the D4 antibody expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. Types of vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. In principle, any vector may be used as long as it is replicable and stable in the host. In addition to the origin of replication, the expression vector may contain a marker gene and other translational regulatory elements.

The invention also includes a host cell comprising a nucleic acid molecule or expression vector as described above.

The host cell expressing the D4 antibody can be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, Streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells or Bowes melanoma cells.

The invention also includes a method for detecting the level of the novel coronavirus SARS-CoV-2 for non-diagnostic purposes, comprising the steps of:

extracting a sample containing new coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with the monoclonal antibody D4;

and thirdly, detecting the immunoreaction of the sample and the monoclonal antibody.

The invention also includes the application of the monoclonal antibody aiming at the non-RBD region of the spinous process protein of the new coronavirus SARS-CoV-2 in the preparation of a detection product of the new coronavirus SARS-CoV-2.

The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product capable of detecting SARS-CoV-2 comprising a binding molecule as described above is included within the scope of the invention.

The invention also includes the application of the monoclonal antibody aiming at the non-RBD region of the spike protein of the new coronavirus SARS-CoV-2 in the preparation of the medicine for inhibiting the new coronavirus SARS-CoV-2.

The invention also includes the application of the monoclonal antibody aiming at the non-RBD region of the spike protein of the new coronavirus SARS-CoV-2 in the preparation of a pharmaceutical preparation for preventing or treating pneumonia caused by the new coronavirus SARS-CoV-2.

The terms "new coronavirus SARS-CoV-2" and "SARS-CoV-2 virus", "new coronavirus", "SARS-CoV-2" and "new coronavirus SARS-CoV-2" used in the present invention can be used interchangeably.

Compared with the prior art, the invention has the following advantages:

the monoclonal antibody aiming at the non-RBD region of the spinous process protein of the new coronavirus has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the region outside the RBD region of the spinous process protein on the surface of the SARS-CoV-2 of the new coronavirus, is used for qualitative or quantitative detection of the new coronavirus, can neutralize and weaken certain toxicity of the new coronavirus, and achieves the purpose of preventing or/and treating pneumonia of the new coronavirus.

The phage display technology inserts exogenous DNA into the gene of phage coding coat protein, so that the expression product corresponding to the exogenous DNA fragment is fused in the coat protein of the phage to form fusion protein, and the fusion protein is displayed on the surface of the phage. Has the following remarkable advantages: direct physical connection between the genotype and the phenotype is established, so that the screening is simple, convenient and efficient. The invention screens the antibody which can be combined with the S protein of the new coronavirus SARS-CoV-2 from the synthetic antibody library Tomlinson I + J phage display antibody library, and the antibodies have important application value in the aspects of detecting the new coronavirus and weakening the virus toxicity.

Drawings

FIG. 1 shows the results of enzyme-linked immunoassay for the antigen binding performance of phages obtained by panning an antibody library;

FIG. 2 shows the results of ELISA assay for the antigen specificity of monoclonal antibody D4;

FIG. 3 shows the results of ELISA assay for the antigen specificity of monoclonal antibody A2;

FIG. 4 shows the results of ELISA assay for the antigen specificity of monoclonal antibody C3;

FIG. 5 is a schematic diagram of the principle of detecting new coronavirus using elutriated antibody;

FIG. 6 shows the results of ELISA using Fab fragment of D4 antibody in combination with A2 to detect virus S protein;

FIG. 7 shows the results of ELISA using Fab fragment of D4 antibody in combination with C3 to detect virus S protein.

Detailed Description

The present invention aims at providing monoclonal antibody against non-RBD region of spinous process protein of new coronavirus and its application, and the present invention is further illustrated by the following examples. The examples of the invention are intended to be illustrative and not limiting, and simple modifications thereof in accordance with the principles of the invention are intended to be within the scope of the claims. The invention is further described with reference to specific examples.

Example 1

Amplification of a phage display antibody library:

mu.L of E.coli TG-1 (MRC HGMP resources center, UK) containing Tomlinson I + J phage display antibody library was inoculated into 25mL of 2YT medium (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) containing 100. mu.g/mL ampicillin and 1% glucose, and cultured at 37 ℃ to OD₆₀₀At 0.4, add 10⁹cfu (Colony Formation Unit) KM13 helper phage (MRC HGMP resources center, UK), 1 hour after infection at 37 ℃, 3000g was centrifuged for 30 minutes, the supernatant was discarded, and 50mL (1.6% Tryptone, 1% Yeast Extract, 0.5%) of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was usedNaCl) suspension bacteria, shaking the bacteria for 16 hours at the speed of 30 ℃ and 250rpm, centrifuging the culture solution for 5000g and 30 minutes the next day, separating and recovering 40mL of supernatant, adding 10mL of PEG/NaCl solution into the supernatant, mixing uniformly, standing on ice for 30 minutes, centrifuging for 5000g and 30 minutes, discarding the supernatant, adding 2mL of sterilized PBS solution to dissolve the precipitate to be used as phage display antibody library solution, titrating the phage display antibody library by using escherichia coli, and obtaining the antibody library with the concentration of 10¹²cfu/mL。

Second, panning of phage display antibody library

mu.L of a PBS solution containing 10. mu.g/ml of SARS-CoV-2 virus S protein (Nanjing Kingkumquat Biotech Co., Ltd.) was added to each of 10 wells of a 96-well microplate, overnight incubation was performed at 4 ℃ and the antigen solution was discarded the next time, 200. mu.L of a PBS solution containing 2% skim milk powder was added to each well, incubation was performed at 25 ℃ for 2 hours for blocking, and after 3 times of PBST washing, 100. mu.L of a phage solution (R0; each well containing 10. mu.L of the antigen solution) was added to each well⁹cfu phage) were incubated at room temperature for 2 hours, and after washing with PBST, phage bound to viral S protein were eluted by adding 100 μ L trypsin per well.

Culturing TG-1 E.coli to OD₆₀₀To 0.4, 4 mL of bacterial solution was taken, 500. mu.L of the dissolved phage solution was added to the bacterial solution, infected at 37 ℃ for 30 minutes, centrifuged at 5000g for 20 minutes, the supernatant was discarded, the cells were suspended in 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose, and shaken at 30 ℃ and 250rpm for 16 hours; centrifuging the culture solution for 5000g and 30 min the next day, separating and recovering the supernatant, adding 1/5 volumes of PEG/NaCl solution into the supernatant solution, uniformly mixing, placing on ice for 30 min, centrifuging for 5000g and 30 min, discarding the supernatant, and adding 200 μ L of sterilized PBS solution as phage solution (R1) after the first enrichment; repeating the steps to respectively obtain phage solutions R2 and R3; and performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of the phage display antibody library and the S protein obtained in the panning process.

The enzyme-linked immunosorbent assay was performed as follows: adding 100 mu L of solution (2 mu g/mL) containing the S protein of the new coronavirus into a 96-well enzyme label plate or addingBovine serum albumin BSA (2. mu.g/mL) in PBS was added overnight at 4 ℃ and the antigen solution was discarded the next day, 200. mu.L of a solution containing 2% skim milk powder was added, incubated at 25 ℃ for 2 hours, and the plate was blocked. The microplate was washed 3 times with PBS containing 0.1% Tween 20, and diluted R0, R2 and R3 phage solutions (10) were added⁹cfu/well), incubation at 25 ℃ for 1 hour, washing the microplate with PBST solution, addition of HRP-labeled mouse anti-M13 antibody, incubation for 1 hour, washing the plate with PBST, addition of HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H₂O₂) And after color development, measuring the absorbance at 450nm by using an enzyme-labeling instrument, drawing a histogram, and comparing the binding performance of the phage antibody, the S protein and the BSA obtained in each step.

The results of the enzyme-linked immunosorbent assay are shown in fig. 1, and when the binding capacities of the phage libraries R0, R2 and R3 obtained in the phage panning process and the S protein are compared, it is found that the binding capacity of the phage solution R2 obtained in the second panning process and the S protein is significantly increased, and the binding performance to BSA is very weak and unchanged, which indicates that the antibodies against the new coronavirus S protein in the constructed phage display antibody library are enriched.

Thirdly, screening of monoclonal antibody

Culturing TG-1 E.coli to OD₆₀₀Taking 100 mu L of elutriation sieve R2 phage antibody library dissolved out phage solution for infecting 200 mu L of escherichia coli bacterial solution, incubating for 30 minutes at 37 ℃, coating the bacterial solution on a 2YT culture medium plate containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 1% glucose, and culturing overnight at 37 ℃; the next day, 96 colonies were picked and inoculated onto 96-well plates, and cultured at 37 ℃ to OD₆₀₀To 0.4, M13 phage was added to each well, centrifuged at 5000g for 20 minutes after infection, the supernatant was removed, 200. mu.L of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was added to each well, the cells were suspended, and cultured at 30 ℃ and 250rpm for 16 hours; centrifuging the culture solution at 5000g for 30 min the next day, separating and recovering the supernatant, performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of each monoclonal antibody and S protein.

Enzyme linkedThe immunoadsorption assay was performed as follows: 100 μ L of PBS solution containing virus S protein (1 μ g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200 μ L of a solution containing 2% skim milk powder was added, incubation was performed at 25 ℃ for 2 hours, and the plate was blocked. Washing the ELISA plate with PBS solution containing 0.1% Tween 20 for 3 times, adding phage solution, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding HRP-labeled mouse anti-M13 antibody, incubating for 1 hr, washing the plate with PBST, adding HRP substrate TMBZ (prepared with sodium acetate solution of pH6.0, containing 1/10000 diluted 30% H)₂O₂) After the development, absorbance at 450nm and at 630nm was measured with a microplate reader, a histogram was plotted, and the binding properties of the phage antibody prepared by each clone to S protein and bovine serum albumin were compared.

Experiments show that 6 micropores are dark in color, the absorbance of the 6 micropores is 1.40, 1.30, 1.35, 1.05, 1.10 and 0.80 respectively, after thalli of the corresponding micropores are subjected to amplification culture, plasmids are extracted from the thalli in the micropores with the absorbance of 0.80 and subjected to gene sequencing, and an antibody is named as a monoclonal antibody D4 according to the position (4 columns on row D) of the antibody in the micropore plate; extracting plasmids from thalli in the micropore with the absorbance of 1.4, carrying out gene sequencing, and naming the antibody as a monoclonal antibody A2 according to the position of the antibody in the micropore plate (A row and 2 columns); extracting plasmids from thalli in the micropore with the absorbance of 1.1, carrying out gene sequencing, and naming the antibody as a monoclonal antibody C3 according to the position of the antibody in the micropore plate (row C and column 3); by aligning with the antibody sequences registered in the antibody gene library, no sequence identical to the antibody gene of the present invention is found, and therefore these antibodies are novel antibodies. The details of the amino acid sequence of the antibody are as follows.

The variable region sequence of the heavy chain of the D4 antibody is SEQ ID NO: 1, CDRH1 sequence of SEQ ID NO: 2; the CDRH2 sequence is SEQ ID NO: 3; the CDRH3 sequence is SEQ ID NO: 4;

the variable region sequence of the light chain of the D4 antibody is SEQ ID NO: 5, CDRL1 sequence is SEQ ID NO: 6; the CDRL2 sequence is SEQ ID NO: 7; the CDRL3 sequence is SEQ ID NO: 8.

the heavy chain variable region sequence of the A2 antibody is SEQ ID NO: 9, CDRH1 sequence as SEQ ID NO: 10; the CDRH2 sequence is SEQ ID NO: 11; the CDRH3 sequence is SEQ ID NO: 12;

the variable region sequence of the light chain of the A2 antibody is SEQ ID NO: 13, CDRL1 sequence of SEQ ID NO: 14; the CDRL2 sequence is SEQ ID NO: 15; the CDRL3 sequence is SEQ ID NO: 16.

the heavy chain variable region sequence of the C3 antibody is SEQ ID NO: 17, CDRH1 sequence of SEQ ID NO: 18; the CDRH2 sequence is SEQ ID NO: 19; the CDRH3 sequence is SEQ ID NO: 20;

the variable region sequence of the light chain of the C3 antibody is SEQ ID NO: 21, CDRL1 sequence of SEQ ID NO: 22; the CDRL2 sequence is SEQ ID NO: 23; the CDRL3 sequence is SEQ ID NO: 24.

the sequence specific information related to D4 of the present invention is as follows:

SEQ ID NO：1：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISYGGNTTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSSGFDYWGQGTLVTVSS；

SEQ ID NO：2：GFTFSSYA；

SEQ ID NO：3：ISYGGNTT；

SEQ ID NO：4：AKTSSGFDY；

SEQ ID NO：5：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAADYPSTFGQGTKVEIKR；

SEQ ID NO：6：QSISSY；

SEQ ID NO：7：NAS；

SEQ ID NO：8：QQAADYPST；

the sequence specific information related to the A2 of the invention is as follows:

SEQ ID NO：9：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIDASGYYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDAYTFDYWGQGTLVTVSS；

SEQ ID NO：10：

GFTFSSYA；

SEQ ID NO：11：

IDASGYYT；

SEQ ID NO：12：

AKDAYTFDY；

SEQ ID NO：13：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASADPDTFGQGTKVEIKR；

SEQ ID NO：14：

QSISSY；

SEQ ID NO：15：

SAS；

SEQ ID NO：16：

QQASADPDT。

the sequence specific information related to the invention C3 is as follows:

SEQ ID NO：17：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIASSGYYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDADSFDYWGQGTLVTVSS；

SEQ ID NO：18：GFTFSSYA；

SEQ ID NO：19：IASSGYYT；

SEQ ID NO：20：AKDADSFDY；

SEQ ID NO：21：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSAPSTFGQGTKVEIKR；

SEQ ID NO：22：QSISSY；

SEQ ID NO：23：AAS；

SEQ ID NO：24：QQAYSAPST。

fourth, antigen specificity of monoclonal antibody

Adding 100 mu L of new coronavirus S protein, S-RBD protein and BSA protein solution with the concentration of 1 mu g/mL into a 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the protein solution the next day, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the enzyme label plate; after washing the microplate 3 times with PBS containing 0.1% Tween 20, 100. mu.L of diluted phage display antibody solution was added to each well, incubated at 25 ℃ for 1 hour, the microplate was washed with PBST solution, HRP-labeled mouse anti-M13 antibody was added, and after incubation for 1 hour, the microplate was washed with PBST, and HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H) was added₂O₂) After the development, absorbance at 450nm was measured with a microplate reader, a histogram was plotted, and the binding properties of the phage antibody produced by each clone and the envelope protein were compared.

The results of ELISA using monoclonal antibody D4 are shown in FIG. 2, in which D4 binds to S protein, but binds to S-RBD protein only weakly and mainly to the region other than the RBD region of S protein, and in which monoclonal antibody D4 binds to coated BSA, indicating that the binding of the antibody to S protein is specific.

The results of ELISA using monoclonal antibody A2 are shown in FIG. 3, in which A2 binds to S protein and S-RBD protein, and in which A2 antibody was compared with coated BSA as an antibody recognizing RBD region of S protein, indicating that the binding of monoclonal antibody A2 to S protein is specific.

The results of ELISA using monoclonal antibody C3 are shown in FIG. 4, in which C3 binds to S protein and S-RBD protein, and in which C3 antibody was compared with coated BSA as an antibody recognizing the RBD region of S protein, indicating that the binding of monoclonal antibody C3 to S protein was specific.

Fifthly, detecting the novel coronavirus S protein by using the Fab fragment of the A2 antibody and the phage-displayed D4 antibody

The detection principle is shown in FIG. 5, the protein A2 and phage display D4 are used for detecting the S protein of the new coronavirus, Fab fragment of A2 antibody is coated in the hole of a 96-hole micropore plate (the heavy chain variable region gene and the light chain variable region gene of A2 antibody are cloned to pDeng 1 phage display carrier, transformed Escherichia coli HB2151 is transformed, the transformed Escherichia coli is cultured, then culture supernatant is taken, histidine tag is used for purification to obtain Fab fragment of A2 antibody), after the micropore plate is sealed, S protein or BSA protein is added, phage display D4 antibody is added after washing the plate, then anti-phage antibody marked with horse radish peroxidase is added, substrate is added after washing the plate for color development, when the sample has no new coronavirus S protein or BSA protein, the reaction system does not develop color, when virus S protein exists, and the more virus S protein exists in the sample, the more D4 of the virus S protein enzyme plate is captured, the more anti-phage antibody captured correspondingly, the darker the color of the enzyme-added substrate after development, and the method can be used to determine whether the sample contains virus S1 protein, and can also be used to detect whether SARS-CoV-2 virus exists in the sample.

The specific operation is as follows: adding A2 antibody Fab fragment into the hole of 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the liquid in the hole, adding 200 μ L of 2% skimmed milk powder solution, standing at room temperature for two hours, and sealing the enzyme label plate. After washing the plate, 100. mu.L of a solution containing the S protein of the novel coronavirus or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25 ℃ for 1 hour, the well-containing solution was removed, the plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed D4 antibody solution (10) was added⁹cfu/mL), incubation for 1 hour at 25 ℃, adding an anti-phage antibody solution (1 mu g/mL) marked with horseradish peroxidase (HRP) after plate washing, incubation for 1 hour at 25 ℃, removing the solution in the wells, washing the plates for 3 times by PBST, finally adding an HRP substrate 3,3,5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution for color development, measuring the absorbance of the solution in the wells at 450nm and 630nm, making a histogram, and comparing the binding capacity of the antibody with the S protein of the new coronavirus and the bovine serum albumin.

As shown in FIG. 6, the results of detecting the virus S protein by the combination of the A2 antibody and phage-displayed D4 were shown in which the horizontal axis represents the name of the detection protein and the vertical axis represents the absorbance of the solution in the wells corresponding to the enzyme. The absorbance of the wells containing the virus S protein enzyme-labeled plate in the solution of the combination of A2 and D4 is 0.47, and the absorbance of the wells added with BSA is 0.04, which indicates that the combination of A2 and D4 can be used for detecting the new coronavirus S protein and the new coronavirus in the solution.

Sixthly, detecting the novel coronavirus S protein by using the Fab fragment of the C3 antibody and the D4 antibody displayed by phage

The detection principle is shown in FIG. 5, C3 protein and phage-displayed D4 are used for detecting S protein of new coronavirus, Fab fragment of C3 antibody is coated in the hole of a 96-hole micropore plate (heavy chain variable region gene and light chain variable region gene of C3 antibody are cloned to pDeng 1 phage display carrier, transformed Escherichia coli HB2151 is transformed, transformed Escherichia coli is cultured and then culture supernatant is taken, histidine tag is used for purification to obtain Fab fragment of C3 antibody), S protein or BSA protein is added after the micropore plate is sealed, phage-displayed D4 antibody is added after the plate is washed, then anti-phage antibody marked with horse radish peroxidase is added, substrate is added after the plate is washed for color development, when the sample has no S protein of new coronavirus or BSA protein, the reaction system does not develop color, when the system develops color when the S protein exists, and the more virus S protein exists in the sample, the more D4 phage is used for enzyme labeling the plate with virus S protein, the more anti-phage antibody captured correspondingly, the darker the color of the enzyme-added substrate after development, and the method can be used to determine whether the sample contains virus S1 protein, and can also be used to detect whether SARS-CoV-2 virus exists in the sample.

The specific operation is as follows: adding the Fab fragment of the C3 antibody into the hole of the 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the liquid in the hole the next time, adding 200 mu L of 2% skimmed milk powder solution, standing for two hours at room temperature, and sealing the enzyme label plate. After washing the plate, 100. mu.L of a solution containing the S protein of the novel coronavirus or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25 ℃ for 1 hour, the well-containing solution was removed, the plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed A3 antibody solution (10) was added⁹cfu/mL), incubation for 1 hour at 25 ℃, adding an anti-phage antibody solution (1 mu g/mL) marked with horseradish peroxidase (HRP) after plate washing, incubation for 1 hour at 25 ℃, removing the solution in the wells, washing the plates for 3 times by PBST, finally adding an HRP substrate 3,3,5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution for color development, measuring the absorbance of the solution in the wells at 450nm and 630nm, making a histogram, and comparing the binding capacity of the antibody with the S protein of the new coronavirus and the bovine serum albumin.

As shown in FIG. 7, the results of detecting the virus S protein by the combination of the C3 antibody and phage-displayed D4 were shown, in which the horizontal axis represents the name of the detection protein and the vertical axis represents the absorbance of the solution in the wells corresponding to the enzyme. The absorbance of the wells containing the viral S protein enzyme-labeled plate in the solution of the combination of C3 and D4 was 0.49, while the absorbance of the wells added with BSA was 0.04, indicating that the combination with D4 can be used for detecting the new coronavirus S protein and new coronavirus in the solution.

Sequence listing

<110> Weifang medical college

<120> monoclonal antibody aiming at non-RBD region of new coronavirus spike protein and application thereof

<130> 20200904A-5

<141> 2020-09-04

<150> 2020104148681

<151> 2020-05-15

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Tyr Gly Gly Asn Thr Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Ser Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Ser Tyr Gly Gly Asn Thr Thr

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Lys Thr Ser Ser Gly Phe Asp Tyr

1 5

<210> 5

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ala Asp Tyr Pro Ser

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 6

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Ser Ile Ser Ser Tyr

1 5

<210> 7

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Asn Ala Ser

1

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Gln Ala Ala Asp Tyr Pro Ser Thr

1 5

<210> 9

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Ala Ser Gly Tyr Tyr Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ala Tyr Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ile Asp Ala Ser Gly Tyr Tyr Thr

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Lys Asp Ala Tyr Thr Phe Asp Tyr

1 5

<210> 13

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Ala Asp Pro Asp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gln Ser Ile Ser Ser Tyr

1 5

<210> 15

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ser Ala Ser

1

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Gln Ala Ser Ala Asp Pro Asp Thr

1 5

<210> 17

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ala Ser Ser Gly Tyr Tyr Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ala Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Ile Ala Ser Ser Gly Tyr Tyr Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Ala Lys Asp Ala Asp Ser Phe Asp Tyr

1 5

<210> 21

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Ala Pro Ser

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 22

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gln Ser Ile Ser Ser Tyr

1 5

<210> 23

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Ala Ser

1

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gln Gln Ala Tyr Ser Ala Pro Ser Thr

1 5

Claims

1. A monoclonal antibody directed against non-RBD regions of the spinous process protein of a novel coronavirus which is characterized in that: the monoclonal antibody is specifically combined with a non-RBD region of a new coronavirus spike glycoprotein and comprises complementarity determining regions CDRH1, CDRH2 and CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8.

2. the monoclonal antibody against non-RBD region of the spinous protein of a neocoronavirus according to claim 1, wherein: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO: 5.

3. an isolated nucleic acid molecule, wherein: the nucleic acid molecule encodes the monoclonal antibody of any one of claims 1-2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method for detecting the level of a new coronavirus, for non-diagnostic purposes, comprising: the method comprises the following steps:

extracting a sample containing new coronavirus;

contacting the sample obtained in the step I with the monoclonal antibody of any one of claims 1-2;

7. The use of the monoclonal antibody against non-RBD region of spinous process protein of a novel coronavirus according to any one of claims 1-2 for the preparation of a novel coronavirus detection product.